Gene organization, evolution and expression of the microtubule-associated protein ASAP (MAP9)

International audienceBackground : ASAP is a newly characterized microtubule-associated protein (MAP) essential for propercell-cycling. We have previously shown that expression deregulation of human ASAP results in profounddefects in mitotic spindle formation and mitotic progression leading to aneuploidy, cytokinesis defects and/or cell death. In the present work we analyze the structure and evolution of the ASAP gene, as well as thedomain composition of the encoded protein. Mouse and Xenopus cDNAs were cloned, the tissueexpression characterized and the overexpression profile analyzed. Results : Bona fide ASAP orthologs are found in vertebrates with more distantly related potentialorthologs in invertebrates. This single-copy gene is conserved in mammals where it maps to syntenicchromosomal regions, but is also clearly identified in bird, fish and frog. The human gene is stronglyexpressed in brain and testis as a 2.6 Kb transcript encoding a ~110 KDa protein. The protein containsMAP, MIT-like and THY domains in the C-terminal part indicative of microtubule interaction, while the N-terminal part is more divergent. ASAP is composed of ~42% alpha helical structures, and two main coiled-coil regions have been identified. Different sequence features may suggest a role in DNA damage response. As with human ASAP, the mouse and Xenopus proteins localize to the microtubule network in interphaseand to the mitotic spindle during mitosis. Overexpression of the mouse protein induces mitotic defectssimilar to those observed in human. In situ hybridization in testis localized ASAP to the germ cells, whereasin culture neurons ASAP localized to the cell body and growing neurites. Conclusion : The conservation of ASAP indicated in our results reflects an essential function invertebrates. We have cloned the ASAP orthologs in mouse and Xenopus, two valuable models to studythe function of ASAP. Tissue expression of ASAP revealed a high expression in brain and testis, two tissuesrich in microtubules. ASAP associates to the mitotic spindle and cytoplasmic microtubules, and representsa key factor of mitosis with possible involvement in other cell cycle processes. It may have a role inspermatogenesis and also represents a potential new target for antitumoral drugs. Possible involvement inneuron dynamics also highlights ASAP as a candidate target in neurodegenerative disease

ASAP, une nouvelle protèine du fuseau mitotique (étude d'un partenaire, la kinase Aurora-A et implication durant le cycle cellulaire)

Le laboratoire a caractérisé une nouvelle protéine associée aux microtubules (MAP) interphasique et au fuseau mitotique, appelée ASAP (ASter Associated Protein) ou MAP9. La dérégulation de son expression entraîne de sévères défauts mitotiques : fuseaux anormaux, retard de la progression mitotique avec des défauts de congression et de ségrégation des chromosomes aboutissant à une cytokinèse défectueuse, des cellules aneuploïdes et à la mort cellulaire. ASAP est donc nécessaire à l assemblage du fuseau et au bon déroulement de la mitose. Les mécanismes contrôlant la mitose et l assemblage du fuseau sont très finement régulés par phosphorylation assurées par plusieurs familles de kinases parmi lesquelles la kinase Aurora-A qui recrute et phosphoryle de nombreuses MAPs. Mon projet principal de recherche consistait à déterminer le lien fonctionnel entre ASAP et Aurora-A. J ai montré qu ASAP était phosphorylée par Aurora-A, caractérisé le site majeur de cette phosphorylation in vivo sur la sérine 625 et montré que cette phosphorylation était essentielle à l assemblage du fuseau mitotique et à la progression correcte de la mitose. Par ailleurs, la déplétion d Aurora-A induit la dégradation d ASAP par le protéasome suggérant que cette interaction et/ou phosphorylation est nécessaire à la stabilisation d ASAP. Ce travail a ainsi permis de caractériser le premier partenaire d ASAP, de confirmer son rôle crucial au cours de la mitose et d en déterminer les premiers mécanismes moléculaires. Enfin, la caractérisation de l orthologue de souris et de son expression tissulaire m a permis de valider le modèle murin comme modèle d étude de différentes fonctions biologiques d ASAP.MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

ASAP is a novel substrate of the oncogenic mitotic kinase Aurora-A : phosphorylation on Ser625 is essential to spindle formation and mitosis

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Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity*

Bipolar spindle formation is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, abnormal number and structural organization of centrosomes can lead to loss of spindle bipolarity and genetic integrity. ASAP (aster-associated protein or MAP9) is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. Its phosphorylation by Aurora A is required for spindle assembly and mitosis progression. Here, we show that ASAP is localized to the spindle poles by Polo-like kinase 1 (Plk1) (a mitotic kinase that plays an essential role in centrosome regulation and mitotic spindle assembly) through the γ-TuRC-dependent pathway. We also demonstrate that ASAP is a novel substrate of Plk1 phosphorylation and have identified serine 289 as the major phosphorylation site by Plk1 in vivo. ASAP phosphorylated on serine 289 is localized to centrosomes during mitosis, but this phosphorylation is not required for its Plk1-dependent localization at the spindle poles. We show that phosphorylated ASAP on serine 289 contributes to spindle pole stability in a microtubule-dependent manner. These data reveal a novel function of ASAP in centrosome integrity. Our results highlight dual ASAP regulation by Plk1 and further confirm the importance of ASAP for spindle pole organization, bipolar spindle assembly, and mitosis